Cyclooxygenases-1 and -2 (COX-1 and COX-2) catalyze the committed step in the conversion of arachidonic acid to prostaglandins (PGs) and thromboxane. COX-1 is constitutively expressed in many tissues and appears to play a role in homeostatic functions whereas COX-2 is highly regulated in response to a range of agonists and appears to contribute to pathophysiological responses. Selective inhibition of COX-2 has been exploited for the development of anti-inflammatory compounds with reduced gastrointestinal toxicities. However, the recent recognition of cardiovascular toxicity associated with the clinical use of these agents suggests that the role of COX-2 in normal physiology has not been fully appreciated. The major functional differences between COX-1 and COX-2 have primarily been attributed to their differential expression. However, we have shown that COX-2 is able to oxygenate a range of neutral derivatives of arachidonic acids, including the endocannabinoid, 2-arachidonylglycerol (2-AG). COX-2-dependent oxygenation of 2-AG leads to the formation of a range of glyceryl esters of PGs (PG-Gs) that is nearly as diverse as the PGs themselves. PG-G formation occurs in macrophage populations responding to inflammatory stimuli, and PGE2-G induces Ca2+ mobilization in RAW264.7 cells, supporting the hypothesis that PG-Gs represent a new class of lipid mediators. Here we propose to further exploit the resources of the Research Center for Pharmacology and Drug Toxicology to answer key remaining questions concerning the possible physiological roles of PG-Gs in vivo. We will 1) apply lipidomics and RNAi technology to monitor total lipid changes in macrophages during PG-G synthesis in order to identify the lipid pools that give rise to 2-AG and the specific enzymes that regulate 2-AG formation. 2) perform detailed kinetic studies of the interaction of hydroperoxy glyceryl esters with COX-2 in order to determine if PGG2-G, the immediate product of COX-2 oxygenation of 2-AG, differs from PGG2 in its ability to activate or inactivate the enzyme;3) characterize PGG formation by rabbit renal medullary interstitial cells, which appear to be a potential source of large quantities of PG-Gs that may be important in the regulation of renal function under hypertonic conditions;4) characterize PGE2-G metabolism in the monkey in vivo to identify unique metabolites of PG-Gs that can be used to quantify in vivo biosynthesis of these compounds in humans. These experiments will provide critical information toward the development of a better understanding of the role of PG-Gs in vivo, and will help test the hypothesis that PG-G synthesis represents a unique physiologic function of COX-2.